JP2005343874A - Method for introducing foreign substance to cell, or the like, using inactivated viral envelope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for introducing a foreign substance to a cell or in vivo tissue, which uses an inactivated viral envelope, is simpler and has excellent operability and high introduction efficiency. <P>SOLUTION: This method for introducing the foreign substance to the cell or the in vivo tissue using the inactivated viral envelope comprises (1) a process for bringing the inactivated viral envelope into contact with a surfactant, (2) a process for reducing the concentration of the surfactant in the composition containing the inactivated viral envelope and the surfactant obtained by the process (1), (3) a process for further mixing the composition containing the inactivated viral envelope brought into contact with the surfactant, obtained in the process (2), with the foreign substance and (4) a process for bringing the composition obtained by the process (3) into contact with the cell or in vivo tissue. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for introducing a foreign substance such as a nucleic acid, protein or drug into a cell or a living tissue.

  Many viral and non-viral vectors have been developed to introduce foreign substances such as genes into cells or living tissues. Non-viral methods generally have a problem that the introduction efficiency of genes and the like is lower than some of viral vectors because substances are taken into cells by endocytosis pathway. Therefore, a hybrid vector in which the membrane fusion ability of Sendai virus (Hemagglutinating virus of Japan: HVJ) is imparted to the liposome, that is, a fusion of Sendai virus and liposome (HVJ-liposome) has been proposed. The membrane fusion ability of the virus enables the introduction of genes and the like into the cytoplasm of cultured cells and living tissue cells, and this technique has been frequently used at the animal experiment level worldwide (Patent Document 1, Non-patent Document 1). Patent Documents 1 and 2)

However, in this HVJ-liposome method, in addition to the preparation of the inactivated HVJ envelope, complicated steps such as preparation of the liposome and purification of the HVJ-liposome after fusion were required. A method for directly encapsulating genes and the like has been proposed (Patent Document 2). In the presence of a gene to be introduced into the cell, the inactivated HVJ envelope is freeze-thawed or treated with a surfactant to encapsulate the gene in the envelope. Next, this is a method of introducing a gene or the like into a cell by bringing the envelope into contact with a cell or the like to be introduced. Inactivated HVJ envelopes can be mass-produced. In particular, the method using a surfactant treatment can introduce foreign substances into cells relatively easily and stably, so it can be used to introduce foreign substances into cells. Has been.
In addition, in Patent Document 2, a method for mixing an inactivated virus with a foreign gene in the presence of a surfactant (page 5, line 26 to page 6, line 1) is described. Prior to the mixing step, the method of bringing the surfactant into contact with the inactivated virus is not mentioned, and the effect is not specifically disclosed.

  On the other hand, in the functional analysis of genes and drugs in cells and living tissues, the major issue is how many types of samples can be introduced and processed in a short time, and there is a need for a more efficient and simpler method. It has been.

US Patent 5,631,237 WO01 / 57204 Dzau, V.D. J. et al. et al. Proc. Natl. Acad. Sci. USA, 93, 1412-111425 (1996). Kaneda, Y .; et al. Molecular Medicine Today, 5, 298-303 (1999).

  From the above technical background, it is an object of the present invention to provide a method for introducing a foreign substance into cells or living tissues that uses an inactivated virus envelope, is simpler, has better operability, and has higher introduction efficiency. is there.

  As a result of intensive studies on a method for introducing a foreign substance into a cell or a living tissue using an inactivated virus envelope, the inventors have inactivated prior to the step of mixing the inactivated virus envelope and the foreign substance. Surprisingly, the present invention has been completed by finding that introduction of a foreign substance becomes highly efficient by bringing a virus envelope into contact with a surfactant.

That is, the present invention provides the following.
(1) A method for introducing a foreign substance into a cell or a living tissue using an inactivated virus envelope, comprising:
1) contacting the inactivated virus envelope with a surfactant;
2) A step of reducing the concentration of the surfactant in the composition comprising the inactivated virus envelope obtained in the step 1) and the surfactant,
3) a step of further mixing a foreign substance into the composition containing an inactivated virus envelope brought into contact with the surfactant obtained in the step 2);
4) A method for introducing a foreign substance comprising a step of bringing the composition obtained in the step 3) into contact with a cell or a living tissue.

(2) The method for introducing a foreign substance according to (1), wherein the step of reducing the surfactant concentration in the composition containing the inactivated virus envelope and the surfactant is by dilution.

(3) Introduction of foreign substance according to (1), wherein the step of reducing the concentration of the surfactant in the composition containing the inactivated virus envelope and the surfactant is by exchanging the supernatant by centrifugation. Method.
In or after the step of mixing a foreign substance into the inactivated virus envelope contacted with the surfactant, the concentration of the surfactant in the composition can be further reduced. In this case, the concentration of the surfactant can be lowered by dilution by adding an aqueous solution such as water or a buffer solution. Accordingly, the present invention also provides the following.

(4) The step of further reducing the concentration of the surfactant in the composition by dilution in the step of further mixing the foreign substance with the inactivated virus envelope contacted with the surfactant, or thereafter (1) A method for introducing a foreign substance according to any one of (3).
The step (4) of (1) above, that is, the step of bringing the composition obtained in the step of further mixing a foreign substance into the inactivated virus envelope contacted with the surfactant and the cell or living tissue is necessary. Depending on the conditions, it may be carried out in the presence of protamine sulfate. Accordingly, the present invention also provides the following.

(5) The step of bringing the composition obtained in the step of further mixing a foreign substance into the inactivated virus envelope contacted with the surfactant and the cell or living tissue in the presence of protamine sulfate (1) The method for introducing a foreign substance according to any one of to (4).

(6) The method for introducing a foreign substance according to any one of (1) to (5), wherein the inactivated virus envelope is an inactivated virus envelope of a virus of the Paramyxoviridae family.

(7) The foreign substance introduction method according to any one of (1) to (5), wherein the inactivated virus envelope is an inactivated HVJ envelope.

(8) The method for introducing a foreign substance according to any one of (1) to (7), wherein the foreign substance is a nucleic acid.

  Compared with the methods for introducing foreign substances (for example, genes) into cells and the like using inactivated virus envelopes reported so far, the method of the present invention has a remarkably high introduction efficiency. In addition, there is no centrifugation step after the step of bringing the inactivated virus envelope into contact with the foreign substance, and the operability has been improved so that the number of foreign substance samples to be introduced can be reduced. Even when the number is extremely large, a composition for introducing a foreign substance can be easily prepared. Therefore, the method of the present invention has increased applicability to analysis of a minute amount sample or high-throughput analysis related to intracellular functions of nucleic acids, proteins, drugs and the like.

  The virus used in the present invention can be any virus as long as it has an envelope having a membrane fusion activity with respect to the cell membrane. Paramyxoviridae, Orthomyxoviridae, Herpesviridae, Hepadna Viridae or Flaviviridae viruses are desirable, more desirably Paramyxoviridae viruses, and more desirably HVJ. In the present invention, the inactivated virus envelope includes these inactivated viruses and / or their envelopes. The virus used in the present invention is propagated in fertilized eggs such as chickens, or is propagated by using a continuous infection system (adding a hydrolase such as trypsin to the culture solution) to mammalian cultured cells and tissues. be able to. In addition, the cloned genomes of these viruses can be used in the present invention for infecting cultured cells to cause persistent infection and proliferating them, mutants thereof, and recombinants thereof.

  In the present invention, the term “inactivation” means that when a virus is mentioned, the gene is inactivated, and the inactivated virus does not replicate the gene and has the ability to grow and infect. I lost it. Examples of virus inactivation methods include UV irradiation, radiation irradiation, and treatment with an alkylating agent, but UV irradiation and treatment with an alkylating agent are desirable. Specifically, the method described in WO01 / 57204 is used. It can be done according to this. The treatment with an alkylating agent is more desirable.

  In the present invention, the “envelope” of a virus is a membrane structure based on a lipid bilayer surrounding the nucleocapsid present in a specific virus having an envelope. The envelope is generally composed of a microprojection structure composed of a spike protein encoded by a viral gene and a lipid derived from the host, and itself has no proliferation ability.

  The inactivated virus envelope used in the present invention can be purified by, for example, ultracentrifugation or column chromatography. A more preferable purification method is a purification method including ion chromatography. Specifically, the method described in Example 1 (1) of WO03 / 014338 and Example 7 (1) to (5) is used. Can be mentioned. The concentration of the suspension of the inactivated virus envelope used in the present invention is not particularly limited, but is usually OD 0.05 to 7.0, and preferably OD 0.1 to 5.0.

  When the inactivated virus envelope is treated with a surfactant, it is preferably treated at a low temperature, preferably 0 ° C to 25 ° C, more preferably 0 to 4 ° C. In the present invention, the final concentration of the surfactant in the step of bringing the surfactant into contact with the inactivated virus envelope is from 0.001% to 0.5%, more preferably from 0.05% to 0.3%. Add as follows. Under these conditions, aging is performed for several seconds to several hours, preferably 3 to 10 minutes. If it is 4 ° C. or less, the aging time may be extended. Examples of the surfactant used in the present invention include octyl glucoside, triton X-100, CHAPS, and NP-40. More preferably, Triton X-100 is used.

  In the method of the present invention, after the step of bringing the inactivated virus envelope into contact with the surfactant, the concentration of the surfactant in the composition is lowered. As the method, an aqueous solution such as water or a buffer solution is used. And dilution of the supernatant and replacement of the supernatant by centrifugation. The method by dilution can be performed prior to the step of mixing the foreign substance, but can also be performed simultaneously with the mixing of the foreign substance. On the other hand, the method by centrifugation needs to be performed prior to the step of mixing with a foreign substance. The centrifugation conditions are 3000 to 15,000 × G for 2 minutes or longer, more preferably 10,000 × G for 5 to 20 minutes. After centrifugation, the supernatant was discarded and obtained. The precipitate can be resuspended in PBS (−) (phosphate buffered saline) or a gene transfer medium not containing a surfactant. In the present invention, examples of the liquid for suspending the inactivated virus envelope include water such as distilled water and UF water, and a buffer solution. Examples of the buffer solution include a phosphate buffer solution or a TE buffer solution (Tris-EDTA buffer solution) in the range of pH 7.0 to 8.0. If necessary, this centrifugation operation can be repeated. In the step of reducing the concentration of the surfactant, the concentration of the surfactant may be 5 to 50 times diluted, but is preferably diluted 100 times or more.

  In the step of mixing the inactivated virus envelope contacted with the surfactant and the foreign substance, after mixing the foreign substance, it is performed at a temperature of 0 ° C. to 25 ° C. for several seconds to several hours, preferably 3 to 10 minutes. Aging may be performed, and if it is 4 ° C. or less, the aging time may be further extended. The concentration and mixing ratio are not particularly limited, and can be appropriately selected. After aging, the inactivated virus envelope holds foreign substances and becomes a substance introduction vector.

  In introducing a foreign substance into cultured cells, protamine sulfate is added to the suspension of the substance introduction vector in the presence of protamine sulfate, if necessary, and added to the cultured cells. Protamine sulfate is added so as to be 1 μg / ml to 100 μg / ml, preferably 10 μg / ml to 50 μg / ml. In the case of adding protamine sulfate, after the step of mixing the above-mentioned foreign substances, after aging, it is diluted with PBS (−) or a gene introduction medium so that the gene concentration is 0.1 μg / μl or less. This is to prevent coprecipitation due to protamine sulfate and genes added thereafter.

  In the present invention, the cells into which foreign substances are to be introduced include eukaryotic cells, particularly animal cell systems, and may be either adherent cell systems or suspension cell systems. In addition, it can be applied to a wide range of cell lines, including early cell lines, stem cell lines, fibroblast cell lines, macrophage cell lines, etc., which are generally difficult to introduce cell lines. Under the culture conditions of these cells, the above-mentioned foreign substance introduction vector is added and brought into contact with the cells. In addition, the substance introduction vector of the present invention can be administered to a living body by an in vivo method, or can be administered systemically. That is, it can be administered by an appropriate administration route according to the disease to be treated, the target organ, etc., for example, it can be administered intravenously, artery, subcutaneously, intramuscularly, or kidney, liver, lung, brain, nerve Can be administered directly to the target site of the disease. If it is administered directly to the disease site, it can be treated selectively by organs. After the above-mentioned addition or administration, a foreign substance is introduced into the cells after a certain period of time under normal cell culture conditions or normal breeding conditions. It is also possible to administer by ex vivo method. In this case, according to a conventional method, cells (for example, lymphocytes, hematopoietic stem cells, etc.) of a subject individual such as a mammal are collected and the substance of the present invention is introduced. What is necessary is just to process with a vector and to return the said cell to an object individual | organism | solid after that.

  In the present invention, the foreign substance introduced into the cell or the living tissue is not particularly limited, but drugs, genes and the like are usually used. Specific examples include antibiotics, chemotherapeutic agents, antiallergic agents, cardiovascular agents, anti-inflammatory agents, antirheumatic agents, hormones, vitamins, anti-neoplastic agents, ribocontrast agents, physiologically active proteins, nucleic acids More specifically, as a nucleic acid to be used, a nucleic acid encoding a target protein that can be introduced into a cell to express the target protein, a ribozyme, an antisense nucleic acid, or an RNA that causes RNA interference is expressed. It can be selected from DNA, siRNA, suicide gene, apoptosis-inducing gene and the like.

Preferred embodiments of the present invention include the following.
(A) A method for introducing a foreign substance into cultured cells using an inactivated HVJ envelope, comprising:
a) contacting an inactivated HVJ envelope suspension with a surfactant such that the final concentration of the surfactant is 0.001 to 0.5%;
b) changing the supernatant by centrifugation and resuspending it in an aqueous liquid not containing a surfactant;
c) a step of further mixing a foreign substance into the composition containing an inactivated HVJ envelope brought into contact with the surfactant obtained in the step b);
d) A method for introducing a foreign substance comprising the step of bringing the composition obtained in the step c) into contact with cultured cells.
(B) The foreign substance entry method according to (A), wherein the surfactant is Triton X-100.
(C) The method for introducing a foreign substance according to (A) or (B), wherein the foreign substance is a nucleic acid.
EXAMPLES The present invention will be specifically described below with reference to examples. However, the examples are provided merely for the purpose of explaining the present invention and for reference to specific embodiments thereof. These illustrations are intended to illustrate certain specific embodiments of the invention, but are not meant to limit or limit the scope of the invention disclosed herein. In the present invention, it should be understood that various embodiments based on the idea of the present specification are possible.

Example 1 (Preparation of inactivated HVJ envelope)
The inactivated virus envelope is an inactivated HVJ envelope suspension after buffer exchange obtained by the method according to (1) of Example 1 of WO 03/014438 and (1) to (5) of Example 7. The liquid was used. Briefly, HVJ grown using fertilized chicken eggs was inactivated by treatment with β-propiolactone, followed by a pretreatment step (filter filtration), a concentration step, and then column chromatography (Q Sepharose FF) and a suspension of inactivated HVJ envelope obtained through a purification step including ultrafiltration (tangential flow filter module (A / G Technology UFP-500-E-3A)) was used.

  150 ml of the HVJ envelope suspension was further concentrated to 13 ml using a tangential flow filter module (A / G Technology UFP-500-E-3A). The OD (absorbance at 560 nm) of this suspension was 0.38 after 20-fold dilution.

To this suspension was added an inactivated HVJ envelope suspension by adding a buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl ₂ ) and the above buffer containing 0.5% methylcellulose. The suspension was prepared so as to have an OD of 5.0 (OD when measured by diluting 20 times 0.25) and a methyl cellulose concentration of 0.1%. This HVJ envelope suspension was dispensed at 15 μl each in a 0.5 ml cryopreservation tube, rapidly frozen in liquid nitrogen, and stored at −80 ° C.

Example 2
2 μl of HVJ envelope suspension (OD5.0) prepared by the method described in Example 1 was placed in a 1.5 mL microtube, and 8 μl of PBS (−) was added. Further, 10 μl of 0.4% Triton X-100 / PBS (−) solution was added and centrifuged at 10,000 × G for 15 minutes, and then the supernatant was removed. The obtained precipitate was suspended in 10 μl of PBS (−), and then a pGL3 / TE solution having a DNA concentration of 1 μg / μl (Promega modified firefly luciferase reporter vector pGL3-Control Vector plasmid DNA dissolved in TE buffer) 10 μl Were mixed and allowed to stand on ice for 5 minutes.

Thereafter, 150 μL of PBS (−) and 5 μl of 10 mg / ml protamine sulfate / PBS (−) solution were added to the inactivated HVJ envelope / plasmid mixture and mixed. The obtained mixture is taken and CHO-K1 cells (Chinese hamster ovary cells, ATCC No. CCL-61, purchased from Dainippon Pharmaceutical Co., Ltd.) on a 96-well plate so as to be 10 μL / well (about 1/16 volume) 5 × 10 ³ cells / well, Ham's F12 + 10% FCS 0.1 ml / well, pre-cultured overnight at 37 ° C., 5% CO ₂ ), and cultured at 37 ° C., 5% CO _{2 for} 24 hours. .

  After the culture, the medium was removed, 50 μl of PBS (+) was added per well, then 50 μl of Substrate Solution of Luciferase Assay Kit Luclite (Perkin Elmer) was added to lyse the cells, and 90 μl of luciferase activity (luminescence) Strength) was measured with MICROPLATE SCINTILLATION & LUMINESENCE COUNTER top count (PACKARD) (Table 1).

Example 3
Take 2 μl of the inactivated HVJ envelope suspension (OD5.0) prepared by the method described in Example 1 in a 1.5 mL microtube and add 2 μl of 0.4% Triton X-100 / PBS (−) solution. And mixed and left on ice for 5 minutes. Further, 6 μl of PBS (−) was added and mixed, and then 10 μL of pGL3 / TE solution with a DNA concentration of 1 μg / μl was added and left on ice for 5 minutes. After standing, 150 μL of PBS (−) and 5 μl of a protamine sulfate / PBS (−) solution having a concentration of 10 mg / mL were added to the mixture and mixed. 10 μl (about 1/16 volume) of the obtained mixture was added to CHO-K1 cells in the same manner as in Example 2, and after culturing for 24 hours, luciferase activity (luminescence intensity) was measured (Table 1).

Comparative Example As a comparative example, a conventional method (using a surfactant) was performed. The inactivated HVJ envelope suspension after buffer exchange obtained by the method described in Example 1 (1) of WO03 / 014338 and Example 7 was used. Gene transfer into the cultured cells was performed according to the protocol of GenomOne (registered trademark, manufactured by Ishihara Sangyo Co., Ltd.) which is a commercially available inactivated HVJ envelope.

  40 μl of inactivated HVJ envelope suspension (OD0.25) was placed in a 1.5 mL microtube, centrifuged at 10,000 × G for 5 minutes, and the supernatant was removed. The obtained precipitate was suspended in 10 μl of PBS (−), and 10 μL of a pGL3 / TE solution having a DNA concentration of 1 μg / μL or 4 μg / μL was added and mixed.

Further, 2 μl of 2% Triton X-100 / PBS (−) solution was added to the mixture, mixed, and centrifuged at 10,000 × G for 5 minutes.
The supernatant was removed, and the resulting precipitate was resuspended in 30 μl of PBS (−), and 5 μl of a 10 mg / ml protamine sulfate / PBS (−) solution was added and mixed. 2 μl (about 1/16 volume) of this mixture was added to CHO-K1 cells in the same manner as in Example 2, and luciferase activity (luminescence intensity) was measured.
A comparison with the results obtained in Examples 2 and 3 is shown in Table 1 (the measured values are average values of 3 wells). The gene introduction method of the present invention showed higher introduction efficiency than the conventional method (comparative example).

Claims (8)

A method for introducing a foreign substance into a cell or biological tissue using an inactivated virus envelope, comprising:
1) contacting the inactivated virus envelope with a surfactant;
2) A step of reducing the concentration of the surfactant in the composition comprising the inactivated virus envelope obtained in the step 1) and the surfactant,
3) a step of further mixing a foreign substance into the composition containing an inactivated virus envelope brought into contact with the surfactant obtained in the step 2);
4) A step of bringing the composition obtained in the step 3) into contact with a cell or a living tissue,
A method for introducing foreign substances including The method for introducing a foreign substance according to claim 1, wherein the step of reducing the concentration of the surfactant in the composition containing the inactivated virus envelope and the surfactant is by dilution. The method for introducing a foreign substance according to claim 1, wherein the step of reducing the concentration of the surfactant in the composition containing the inactivated virus envelope and the surfactant is by exchanging the supernatant by centrifugation. The step of further reducing the concentration of the surfactant in the composition by dilution in the step of further mixing with the foreign substance in the inactivated virus envelope contacted with the surfactant, or thereafter. The method for introducing a foreign substance according to any one of 3 above. The method according to any one of claims 1 to 4, wherein the step of bringing the composition obtained in the step of further mixing a foreign substance into the inactivated virus envelope contacted with the surfactant and the cell or living tissue is carried out in the presence of protamine sulfate. A method for introducing a foreign substance according to any one of the above. The method for introducing a foreign substance according to any one of claims 1 to 5, wherein the inactivated virus envelope is an inactivated virus envelope of a Paramyxoviridae virus. The method for introducing a foreign substance according to any one of claims 1 to 5, wherein the inactivated virus envelope is an inactivated HVJ envelope. The foreign substance introduction method according to any one of claims 1 to 7, wherein the foreign substance is a nucleic acid.